The field of this invention is immunocompromised mammals comprising xenogeneic tissues which are capable of long term reconstitution of myeloid and lymphoid cells.
Hematopoiesis is a continuous process of differentiation and amplification, replacing billions of mature lymphoid and myeloid cells in the normal human every day. This process depends on the continuous turnover of hematopoietic stem cells, which have the capacity for self-maintenance, extensive proliferation, and multipotentiality. These characteristics have been studied in great detail in the murine system, particularly through the uses of sequential bone marrow transplants and genetic marking using chromosomal rearrangements or retroviruses. Similar studies with human hematopoietic stem cells have lagged, primarily due to a lack of an equivalent model for long-term multipotential differentiation.
Scientists have recently succeeded in demonstrating human hematopoietic progenitor engraftment and differentiation in immunodeficient mice. Of particular interest has been the use of such mice for studying the tissue, its response to drugs and changes in the environment of the tissue. Various aspects of the human tissue may be studied in an environment simulating the natural environment using such chimeric animals.
Significant advances have been made in understanding the earliest events in human hematopoietic development by transplanting human cells or tissues into immunocompromised mice and observing human hematopoiesis for prolonged periods. However, each of the prior art systems is limited in its ability to study concomitant human mature T-cell, mature B-cell and myeloid cell production from a common stem cell pool. Implants of human fetal thymus and liver are limited in the extent to which myeloid and B-cells will develop. It is therefore of interest to develop a chimeric animal which is capable of long term reconstitution of myeloid, as well as both B- and T-lineages of the hematopoietic system.
A description of the SCID-hu mouse may be found in J. M. McCune et al. (1988) Science 241:1632-1639; R. Namikawa et al. (1990) J. Exp. Med. 172:1055-1063 and J. M. McCune et al. (1991) Ann. Rev. Immunol. 9:395-429. Implantation of functional bone marrow is described in S. Kyoizumi et al. (1992) Blood 79:1704. European patent application no. 469 632 discloses the use of immunocompromised mammals with a thy-liv implant.
Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in SCID mice is described in T. Lapidot et al. (1992) Science 255:1137; J. Nolta et al. (1994) Blood 83:3041; and S. Kyoizumi et al (1993) Blood 81:1479-1488. The proliferation and engraftment of immature cord blood progenitors in such mice is further discussed in J. Vormeer et al. (1994) Blood 83:2489.
Immunocompromised mouse strains are described in S. Nonoyama et al. (1993) J. Immunol 150:3817-3824; I. Gerling et al. (1994) Diabetes 43:433-440; Bosma, et al. (1983) Nature 301:52; and P. Mombaerts et al. (1992) Cell 68:869-877.
Immunocompromised hosts are provided, comprising xenogeneic functioning hematolymphoid tissue comprising bone marrown spleen and optionally, thymus tissue. The tissue grows to form a hybrid organ structure capable of producing B-lineage lymphocytes, T-lineage lymphocytes and myeloid cells.
Methods and compositions are provided for the production of human hematopoietic cells with a plurality of lineages in an immunocompromised heterologous mammalian host, particularly a mouse, for extended periods of time. The method comprises combining non-dispersed bone and spleen fragments in juxtaposition, optionally together with a thymus fragment, in an appropriate site in an immunocompromised host. The chimeric animal is useful for studying human hematopoiesis and pathogenesis in an experimental setting.
The co-implantation of human bone and spleen tissue is sufficient to support the growth of hematopoetic progenitors which, in the absence of a thymus implant, are able to mature into myeloid lineage cells as evidenced by expression of CD33, including granulocytes, as evidenced by the expression of CD14 and CD15; monocytes; and B lineage cells, as evidenced by the expression of CD19 and CD20. Myeloid cells may include neutrophils, monocytes and macrophages, eosinophils, basophils and mast cells, and progenitors thereof. Some T cell subsets are also present in the bone and spleen implant, as shown by the expression of CD4. However, maturation of T cell progenitors to provide all T cell subsets, particularly those that express CD8, requires the presence of a co-implantation of thymus, which provides stromal and epithelial cells necessary for differentiation. When thymus tissue is present, a subset of progenitor cells derived from the bone/spleen co-implant are able to differentiate into T cells, including CD4+CD8+, CD4+CD8xe2x88x92 and CD4xe2x88x92CD8+ subsets.
The host animal is engrafted with both spleen and bone, where the tissue implants are normally contiguous to provide a continuous source of hematopoietic progenitor cells. The spleen tissue appears to amplify to partially or wholly surround the growing human fetal bone and thymus to form a hybrid tissue, which provides a continuous source of myeloid cells, B-cells and other lymphoid progenitor cells. To provide for maturation of T cells, human thymus is also engrafted, in close proximity, usually in contact with the bone and spleen tissue. The thymus tissue may have a different HLA allotype from the spleen and bone. Differences in HLA have shown that mature T-cells are derived from stem cells present in the spleen/bone graft. Such animals are useful in determining the contribution that thymic stromal and epithelial elements, or hematopoietic progenitor cells make to T-cell maturation. The hybrid organ, containing bone, thymus and spleen (BTS), is vascularized, and able to survive in the host for long periods of time. The hybrid tissue may be used after at least about 3 weeks, more usually after at least about 6 weeks, and the hybrid tissue will remain functional for at least about 9 months, or more.
A suitable site for implantation must be able to accomodate the size of the implanted tissue and to keep the implanted tissues in close proximity. Of particular interest is subcutaneous implantation. The position of the subcutaneous implant on the body of the host is not critical, but the area of the mammary fat pads may conveniently be used. The tissue will be implanted, conveniently by incision of the host skin and placement with a trocar, etc.
The BTS tissue transplant may be only one of other tissues transplanted into the host. For example, in addition to the BTS implant, other hematopoietic components may be included, such as stem cells, lymph nodes, embryonic yolk sac, fetal liver, pancreatic tissue, appendix tissue, tonsil tissue and the like, which may serve in the development of a hematopoietic system in the immunocompromised host for a variety of purposes. Sites for introduction of additional tissue may include under the spleen capsule, abdominal wall muscle, under the renal capsule, in the eye, the peritoneum, the peritoneal lining, brain, subcutaneous, vascular system, spinal cord, membranous sacs or capsules of various tissue, the retroperitoneal space, reproductive organs, etc.
Introduction of the secondary tissue may be achieved by injection, implantation, or joining blood vessels (and other vessels if necessary) of the donor and host, using intravenous catheters, trocars, and/or surgical incision, or the like. The tissue or cells of interest will generally be normal, e.g. non-transformed and non-malignant tissue or cells. With various organs one may include native surrounding tissue with the organ tissue itself. The surrounding tissue may comprise connective tissue, or portions of blood and lymphatic vessels. In some cases, whole organ grafts may be transplanted by anastomosing donor and host blood vessels, lymphatic vessels, and the like. For the most part, normal cells, tissue, and/or organs may be stably maintained and functional for at least about 3-6 months and frequently for at least about 10 months.
As appropriate, dispersed cells may be employed, where the relevant organs are teased apart to yield viable cells in suspension. Cells of particular interest as a secondary implant are human hematopoietic cells, particular committed progenitor cells and stem cells. The progenitor cells may be mismatched as to HLA type with the hybrid tissue, so as to provide a marker for differentiating cells. The progenitor cells may be injected into the bone marrow cavity before or after implantation, and the resulting repertoire of lineages analyzed. Ablation of endogenous hematopoietic cells in the bone, e.g. radiation, may be desirable to improve the frequency of engrafted cells. Multilineage stem cell assays can be performed in this manner. The repertoire of lineages which are able to develop from a particular progenitor cell type, and the effects of various treatments of those cells, e.g. exposure to growth factors, cytokines, mutagens, etc. is determined.
A mixed population of cells in suspension may be enriched for the particular cells of interest. For example, with bone marrow cells, the suspension may be enriched for hematopoietic precursors by Ficoll-hypaque density gradient centrifugation, fluorescence activated cell sorting, panning, magnetic bead separation, elutriation within a centrifugal field, or rosetting.
In some instances it may be desirable to enrich cells by killing or removing other cells. This may be achieved by employing monoclonal antibodies specific for the undesired cells in the presence of complement or linked to a cytotoxic agent, such as a toxin, e.g. ricin, abrin, diphtheria toxin, or a radiolabel, e.g. 131I, or the like. Immunoaffinity columns may be employed which allow for specific separation of either the desired or undesired cells, depending on the nature of the mixture.
The human fetal tissue, either BTS or an additional implant, may be fresh tissue, obtained within about 48 hours of death, or freshly frozen tissue, tissue frozen within about 12 hours of death and maintained at below about xe2x88x9210xc2x0 C., usually at about liquid nitrogen temperature (xe2x88x9270xc2x0 C.) indefinitely. The tissue may be from an organ implanted in a chimeric host, where the tissue may be removed from 2-4 weeks after implantation, or longer. In this manner, the tissue originally obtained from the host source may be greatly expanded, substantially increasing the total number of chimeric hosts which may be obtained. The tissue obtained from the chimeric host may be treated analogously to the tissue obtained from the human source. Normally the tissue will not have been subject to culture in vitro for an extended period of time.
The thymus and spleen tissue are provided as pieces of whole organs, and will include such stromal and epithelial cells as are normally present. The size of implanted tissue will generally be from about 0.5 to 4 mm, more usually from about 1 to 2 mm, so that the sections can easily fit into a trocar used for implantation, usually conveniently of about 15- to 20-gauge. The bone marrow may be fetal or adult, preferably fetal. Long bones are employed, such as tibia, femur, humerus or the like. The bone will generally be at least about 0.5 cm in length and may be 2 cm in length or greater, depending upon the size of the host. For a mouse host, 1 cm is found to be a convenient size. The bone may be cut along a longitudinal axis, so that the bone cortex as well as intramedullary regions are exposed to allow for vascularization, or cross-sectional to provide tubular slices.
For the most part the donor tissue will be human, although cells from sources other than members of the same family as the host animal may find use. The source of the tissue will usually be fetal. Preferably the tissue will be from a child of less than about 3 years, preferably less than about 1 year and at or younger than neonate, more preferably being fetal tissue of from about 7 to 24 weeks. In some cases adult human bone may be implanted, as tubular slices or chips.
For different organs differently aged tissue may be preferred. For fetal tissue, it is desirable that the human lymph be equal to or greater than about 20 gestational weeks (g.w.), preferably 20-24 g.w.; and for human thymus and liver, from about 16-24 g.w., preferably greater than 18 g.w. For fetal bones and spleen, the fetus will generally be from about 16 to 24 g.w.
Immunocompromised mammalian hosts suitable for implantation and having the desired immune incapacity exist or can be created. The significant factor is that the immunocompromised host is incapable naturally, or in conjunction with the introduced organs, of mounting an immune response against the xenogeneic tissue or cells. Therefore it is not sufficient that a host be immunocompromised, but that the host may not be able to mount an immune response after grafting, as evidenced by the inability to produce functional syngeneic host B-cells, particularly plasma cells, and/or T-cells, particularly CD4+ and/or CD8+ T-cells after implantation. Of particular interest are small mammals, e.g. rabbits, gerbils, hamsters, guinea pigs, etc., particularly murines, e.g. mouse and rat, which are immunocompromised due to a genetic defect which results in an inability to undergo germline DNA rearrangement at the loci encoding immunoglobulins and T-cell antigen receptors.
Presently available hosts include mice that have been genetically engineered by transgenic disruption to lack the recombinase function associated with RAG-1and/or RAG-2 (e.g. commercially available TIM(trademark) RAG-2 transgenic), to lack Class I and/or Class II MHC antigens (e.g. the commercially available C1D and C2D transgenic strains), or to lack expression of the Bcl-2 proto-oncogene. Of particular interest are mice that have a homozygous mutation at the scid locus, causing a severe combined immunodeficiency which is manifested by a lack of functionally recombined immunoglobulin and T-cell receptor genes. The scid/scid mutation is available or may be bred into a number of different genetic backgrounds, e.g. CB. 17, ICR (outbred), C3H, BALB/c, C57B1/6, AKR, BA, B10, 129, etc. Other mice which are useful as recipients are NOD scid/scid; SGB scid/scid, bh/bh; CB.17 scid/hr; NIH-3 bg/nu/xid and META nu/nu. Transgenic mice, rats and pigs are available which lack functional T cells due to a homozygous disruption in the CD3xcex5 gene. Immunocompromised rats include HsdHan:RNU-rnu; HsdHan:RNU-rnu/+; HsdHan:NZNU-rnu; HsdHan:NZNU-rnu/+; LEW/HanHsd-rnu; LEW/HanHsd-rnu/+; WAG/HanHsd-rnu and WAG/HanHsd-rnu/+. The availability of scid/scid mice with an NOD (non-obese diabetic) background provides an opportunity to study the effect of human T cells in the development of insulin dependent diabetes.
Additional loss of immune function in the host animal may be achieved by decreasing the number of endogenous macrophages before, during, or after implantation of the xenogeneic tissue. Of particular interest is the reduction of macrophages by administration of dichloromethylene diphosphonate (Cl2MDP) encapsulated in liposomes, as described in co-pending application Ser. No. 08/169,293. Elimination of host macrophages improves the ability of non-autologous hematopoietic cells to survive in the host animal""s circulation.
The host will usually be of an age less than about 25% of the normal lifetime of an immunocompetent host, usually about 1 to 20% of the normal lifetime. Generally, the host will be at least about six weeks old and large enough to manipulate for introduction of the donor tissue at the desired site. For example, mice are usually used at about 6 to 10 weeks of age. Growth of the tissue within the host will vary with the organ.
The mammalian host will be grown in conventional ways. Depending on the degree of immunocompromised status of the mammalian host, it may be protected to varying degrees from infection. An aseptic environment is indicated. Prophylactic antibiosis for protection from Pneumocystis infection may be achieved for scid/scid mice with 25-75 mg trimethoprim and 100-300 mg sulfamethoxazole in 5 ml of suspension, given three days each week, or in impregnated food pellets. Alternatively, it may be satisfactory to isolate the potential hosts from other animals in gnotobiotic environments after cesarean derivation. The feeding and maintenance of the chimeric host will for the most part follow gnotobiotic techniques.
The presence of the foreign tissue in. an immunocompromised host may be used to study the effect of various compounds on the growth, viability, differentiation, maturation, transformation, or the like, of the human cells in a live host. The chimeric host may be used to study the effect of a variation of a condition on a symptom or indication of a disease. By condition, it is intended a physical, chemical or biological property, e.g. temperature, electric potential, ionic strength, drugs, transformation, etc.
It is of particular interest to study the pathogenesis of various infectious agents and/or the effect of various drugs or treatments on the induction or progress of disease. Infectious agents of interest include bacteria, such as Pneumococcus, Staphylococcus, Streptococcus, Meningococcus, Gonococcus, Eschericia, Klebsiella, Proteus, Pseudomonas, Salmonella, Shigella, Hemophilus, Yersinia, Listeria, Corynebacterium, Vibrio, Clostridia, Chlamydia, Mycobacterium, Helicobacter and Treponema; protozoan pathogens, and viruses. Viruses of interest include human immunodeficiency viruses (HIV-1 and HIV-2); enteric viruses, e.g. coxsackie, echovirus, reovirus; respiratory viruses, e.g. rhinovirus, adenovirus, coronavirus, parainfluenzavirus, influenzavirus; picornavirus; rhabdovirus; rubeola; poxvirus; herpesvirus; EBV; paramyxovirus (measles), hepatitis viruses A, B, C and D, varicella zoster virus (chicken pox) and cytomegalovirus.
Of particular interest are human tropic viruses, which infect or cause disease in human cells, many of which cannot easily be studied in conventional animal models. In general, human tropic viruses primarily cause productive infections, i.e. infection which results in viral replication and release of new infectious particles, in human cells. The virus may infect cells of closely related primate species, but will usually not cause the disease symptoms seen in humans, e.g. measles virus. The reason for such a tropism to human cells may be due to specific binding of the virus to a particular cell surface antigen required for entry of the virus into the cell. Examples of this specificity are the binding of HWV-1 to human CD4, the binding of herpes simplex-1 to human fibroblast growth factor receptor, and of measles virus to human CD46. Other viruses may require cytoplasmic components of human cells in order to complete a cycle of replication. Human tropic viruses include HIV-1 and HIV-2; the human herpesviruses: HSV-1, HSV-2, varicella zoster virus, Epstein-Barr virus, human B-cell lymphotropic virus and human cytomegalovirus; smallpox virus; measles virus and hepatitis B virus.
The virus may be wild-type, e.g. clinical isolates, conventional strains, etc.; attenuated strains; or may be genetically engineered to enhance or reduce infectivity, pathogenicity, etc. Such modifications in the viral genome may include deletion of virulence genes, mutations in viral coat proteins which alter the host range, change in viral nucleic acid polymerases, alterations in proteins which affect integratation of the viral genome into the host genome, etc. Mutations introduced into the viral genome are useful to map the functions of viral proteins, and to determine which domains are responsible for various aspects of the infection, i.e. in establishing latency, transforming cells, viral replication, etc.
To study the effects of of infection on human cells, a xe2x80x9cBTSxe2x80x9d implant is inoculated with an infectious level of virus. The effect of the virus is determined, usually as a function of time. Data may be obtained as to the immune response of human cells to the virus; products which are secreted by infected or involved cells in response to infection, e.g. cytokines, interferons, antibodies, etc.; the viability and growth of the human lymphocytes, myeloid cells, and stromal cells which are present either in the xe2x80x9cBTSxe2x80x9d implant or in the host circulation; and virus replication, e.g. release of new infectious particles.
Infection may be achieved by direct injection of the virus. Usually, the injection will involve at least about 102 infectious units, preferably from about 103 to 105 infectious units of virus. The virus may be a clinical isolate, a cloned clinical isolate, a genetically modified isolate, or the like. Alternatively, viruses may be administered by injection of infected cells, where the injected cells will produce infectious virus over time. The cells will deliver a dose of virus of at least about 102 infectious units, preferably from about 103 to 105 infectious units of virus.
Various drugs may be administered to the host and the effect on a particular tissue determined by invasive or non-invasive techniques. Non-invasive techniques include NMR, CAT scans, fluoroscopy, roentgenography, radionuclide scanning, ultrasonography, electrocardiography, electroencephalography, evoked potentials, etc. Invasive techniques include biopsy, autopsy, laparotomy, intermittent intravenous blood sampling, or intravenous catheterization, etc. Convenient placement of various devices, e.g. catheters, electrodes, etc. may be performed for continuous monitoring. Thus, the host may be used to determine the carcinogenicity of various compounds to different human tissues, the effect on growth and viability of various human tissues, the effect of combinations of compounds, e.g. drugs, or the like. In addition, by providing for pathogenic infection of the xenogeneic tissue, the effect of various drugs in protecting the host tissue from the pathogen, as well as being cytotoxic to or suppressive of the pathogen in a cellular environment can be determined.
The chimeric hostmay also be used for evaluating the cytotoxicity of various drugs toward human tissue, for example, for screening for investigative new drug applications. In addition, the chimeric hosts may be used for evaluating drugs as to their efficacy, safety and bio-availability. Use of the chimeric animal in studying the effect of drugs on infection may begin with administration of-the drug prior to, substantially concomitant with, or subsequent to the administration of the infectious dose of virus. Administration of the drug will usually begin not earlier than 7 days prior to infection, more usually not more than about 1 day prior to infection. In most cases, administration of the drug will begin not later than about 7 days after infection, more usually not later than about 1 day after infection. However, for studies of chronic infections, drug treatment may be started after as much as. one year after infection, usually after six months, more usually after one month. After initial screening, different periods of time may be of interest in establishing the effectiveness of the drug.
The manner of administration will vary greatly, depending upon the nature of the drug. It may be provided orally, ad libitum, intraperitoneally, intravascularly, subcutaneously, intrathymically, or the like. Usually, different dosage levels will be employed, based on past experience with the drug, anticipated levels with human treatment, toxicity or side effects, experience with the particular chimeric host, and the like. The effect of the drug may be monitored for any convenient time, usually at least 1 week from the initiation of administration of the drug, more usually at least 2 weeks, and at times for periods as long as 6 weeks or more. Preferably, determinations will be made in the period from about 2-6 weeks.
For the effectiveness of drugs in suppressing HIV-induced T-cell or thymocyte depletion, various measurements can be made. By employing flow cytometry (fluorescence-activated cell scanning flow cytometry), one can analyze the CD4 and CD8 profile of the peripheral blood, the cell population in a cell dispersion prepared from the thymus, lymph node implant, or other human fetal tissue which is present, as appropriate. One may also monitor for the presence of HIV, by monitoring the level of p24 in the peripheral blood or the implant, HIV RNA or portion thereof, or HIV DNA, using the polymerase chain reaction. In addition, one may use histological analysis, employing immunochemistry, for detecting the presence of CD4 or CD8, proteins of HIV, e.g. p24, which are present in the implant. One may also analyze for indications of apoptosis in the infected tissue, as indicated by multiple foci of cells with condensed nuclear material as seen by histologic methods or election microscopy or as determined by methods which can discern a DNA degradation profile consistent with apoptosis.
Phenotyping of the xenogeneic cells to verify their origin and stage of developmental progression may be performed by standard histological methods, by immunohistochemistry, antibody staining or in situ hybridization with RNA and/or DNA probes. The exact method is not critical to the invention, and will depend on the exact cell types being studied. HLA markers may be used to distinguish the established xenogeneic organ transplants. The HLA type can be readily determined by staining with an appropriate antibody directed against any of the alleles of the human HLA locus, including Class I and Class II antigens.
The presence of mature human T-cells, B-cells and myeloid cells in an animal model allows the development of human antibodies to a specific antigen. The human cells are able to interact so as to provide T-independent and T-dependent antibody responses, e.g. class-switching from IgM to IgG and IgA subclasses, and affinity maturation of antibody binding. The desired antigen will generally be formulated with an adjuvant. The animals are immunized systemically, e.g. i.v., or intra-muscularly, or intragraft. The animals are boosted with antigen as necessary. A bleed is done on the immunized animals to test the titer of serum antibodies against the immunizing antigen. If production of hybridoma antibodies is desired, the graft will then be removed, and a cell suspension made. The BTS cells are then fused to a myeloma cell partner. The resulting hybridoma cells are screened for reactivity against the immunizing antigen, and positive cells selected for antibody production.